Field of the Invention
The disclosure relates generally to the field of nucleic acid sequence determination and, more specifically, to deep sequencing approaches to parallel sequence determinations of nucleic acids, e.g., small RNAs, microRNAs, piRNAs.
Description of the Relevant Art
Small RNA sequencing (sRNA-seq) is now the gold standard for small RNA profiling and discovery in fields such as the study of biomarkers in cancer, differentiation in stem cells and transposon silencing in the germline. Biases in deep sequencing which affect the profiling have been reported, but their etiology has not been identified.
The advent of deep sequencing has now made it possible to sequence the full complement of small RNAs in a cell. Small RNAs (15-30 nucleotides or nts) play an important role in a variety of cellular processes. MicroRNAs, piRNAs and endogenous siRNAs are among the many small RNAs that are crucial regulators of genetic activity. Small RNA profiling through deep sequencing has become important for understanding the biology of these genes and for identifying miRNA markers for diagnostic and therapeutic uses. Real-time PCR (RT-PCR) and microarrays can be used to profile known small RNAs but a better approach to identifying differences between closely related microRNAs, and to the discovery of novel sequences, is deep sequencing. Subtle variations, such as found in isomers, and modifications, such as uridylation, cannot be detected by microarrays or RT-PCR. In addition, microarrays and RT-PCR also suffer from cross-hybridization artifacts. Deep sequencing is especially attractive for its sensitivity to low abundance transcripts. In light of these benefits of deep sequencing, a persistent mystery in the field of small RNA sequencing is the discrepancy between the results from deep sequencing, microarrays and qPCR.
Accordingly, a need continues to exist in the art for unbiased methods of efficiently and accurately profiling the RNAs of a biological sample such as a cell. A need also continues to exist for deep sequencing techniques that show a reduced, or absent, bias in sequenced RNAs.